DNA nanoparticles self assemble from plasmid DNA and CK30 covalently linked to polyethylene glycol, and effectively transfect airway epithelium, retina, and neurons, but how they access cells has been obscure. Recently the surface receptor for these DNA nanoparticles has been identified as nucleolin. Gene expression from DNA nanoparticles varies directly with the amount of surface nucleolin in cell culture, and the best target tissues in vivo are those with surface nucleolin. How surface expression of nucleolin is regulated, or a protein lacking membrane spanning domain or glycolipid anchor is held at the membrane is unclear. The non-degradative route from cell surface to nucleus followed by the nucleolin/DNA nanoparticle complex is not established. This project will determine the mechanisms by which the DNA nanoparticle receptor, nucleolin, arrives at and is held at the cell surface, as well as the mechanism by which DNA nanoparticles enter the cell and are transported to the nucleus without degradation. The hypotheses are: 1) nucleolin reaches the surface in response to phosphorylation and is held at the surface by interaction with other proteins, some of which are important for DNA nanoparticle binding and uptake 2) the non degradative pathway taken by the nucleolin/DNA nanoparticle complex to the nucleus is susceptible to pharmacologic manipulation. To test these hypotheses, the proteome of DNA nanoparticles and nucleolin at the cell surface will be determined and the importance of the proteins identified tested by siRNA knockout. Fluorescence microscopy, cell fractionation, inhibitor studies, and siRNA analysis will be used to determine the trafficking through the cell. THe phosphorylation state of nucleolin at the cell surface will be tested by mass spectrometry. In this way, a comprehensive picture of the regulation of the route into the cell for DNA nanoparticles will emerge and drug able targets will be suggested. If we are successful, we will improve understanding of pathways into the cell that reach the nucleus without censoring the cargo - pathways which are utilized by pathogens as well as therapeutic agents such as DNA nanoparticles. If we are successful, we will have at our disposal a therapeutic armamentarium to improve gene delivery from DNA nanoparticles. PUBLIC HEALTH RELEVANCE: DNA nanoparticles are promising for gene therapy because they transfect nondividing cells, are non-toxic, non- immunogenic, efficient in some tissues, and can be prepared for high level, long term expression. Manipulating their trafficking to improve transfection improves their versatility, cost-effectiveness, and suitability for human use. In addition, the non-degradative pathway they follow in the cell is probably shared by some pathogens. Further understanding of this route may be important in regulating it. .